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Abstract The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

When reviewing the safety of food packaging, safety assessors must determine the migration levels of a food contact substance (FCS) from the packaging into the contained food products. Migration values can be expressed relative to levels in food using a single ‘food mass-to-surface area (mF/A) ratio’, representative of the general food supply. Because no published literature has examined mF/A ratios for the current U.S. food supply, this study aimed to use food databases to develop a standard mF/A ratio representative of packaged foods in the current U.S. food supply. First, we obtained information on the food-contact surface area (A) and food mass (mF) for a selection of packaged foods sold in U.S. retail stores. To adequately represent the variation of food products on the market, we identified a representative product per food category, using a 95-category list that was derived from the USDA WWEIA Food Categories. The values for A and mF per package were determined for each representative product using product size information from the Syndigo Nutrition Database. These data were used to calculate the A/mF ratio for each of the 95 representative products, and the average A/mF ratio was determined. The average A/mF ratio was weighted using consumption data from the WWEIA component of NHANES, so that more weight was given to foods that are consumed in greater quantities. The final step was to then take the reciprocal of the averaged A/mF ratio to obtain the final mF/A ratio. The resulting mF/A ratio was 4.8 g/in2, which rounds to 5 g/in2. In summary, we determined that 5 g/in2 is representative of the current U.S. food supply and could be used as a standard mF/A ratio when estimating migration levels of an FCS from packaging into food.

The increasing intensity and frequency of climate impacts exacerbate pressures on front-line local communities. This calls for location-specific adaptation strategies. Alignment of strategies with respective National Climate Change Strategy is key for the overall sustainability of initiatives and local communities. The work presented in this paper examines the adoption and prioritization of climate adaptation policies at the local level based on a case study of the Adansi North District (AND) in Ghana. An assessment of the extent to which climate adaptation policies are captured and budgeted for was done via a review of the district’s medium-term development and key political actors were interviewed to assess the level of priority they place on climate adaptation. Findings from the study reveal that 41% of the locally adopted policies directly align with stipulated national level policies. We attribute the adoption of climate policies in AND to local political actors having higher education which has afforded them good understanding of the climate change phenomenon, being experienced professionals and having to work within institutional rubrics that make climate policy formulation a requirement. However, little priority is given to these policies for implementation, mainly through the non-allocation of funds. We account for this with the weak environmental advocacy in the district and exchange between actors on adaptation. Furthermore, partisan actors who already wield veto powers and can promote policies that may not necessarily support adaptation measures, often do so, since their interest is to become popular among electorates who also prefer infrastructure over environmental policies. We conclude that although climate adaptation policies are fairly adopted and budgeted for in AND, they have not received commensurate priority for implementation. Recommendations are proposed for addressing this.

Although corticosteroid therapy is the standard of care for all patients hospitalized with severe coronavirus disease 2019 (COVID-19), the studies demonstrating the mortality–benefit ratio of corticosteroids were limited to fully evaluate their adverse effects. To determine the severity of corticosteroid-induced hyperglycemia in patients with and without diabetes mellitus, we retrospectively collected data from the medical records of patients hospitalized with COVID-19 before and after corticosteroids were the standard of care. Corticosteroid-induced hyperglycemia was more severe in patients hospitalized with COVID-19 with diabetes than those without diabetes. Additionally, patients with diabetes required higher doses of correctional insulin per day when on corticosteroid therapy, suggesting that intensive point-of-care glucose monitoring could be limited in patients without diabetes mellitus and support cautionary use of corticosteroids in patients with COVID-19 discharged with supplemental oxygen.

BackgroundAntiretroviral therapy (ART) decreases perinatal HIV transmission, but concerns exist regarding maternal and infant safety. We compared the incidence of congenital malformations and other adverse outcomes in pregnancies exposed to integrase inhibitor (INSTI) versus non-INSTI ART.SettingSingle-site review of all pregnancies among women living with HIV between 2008 and 2018.MethodsWe used binomial family generalized estimating equations to model the relationship of congenital anomalies and pregnancy outcomes with exposure to INSTI or dolutegravir (DTG) versus non-INSTI ART.ResultsAmong 257 pregnancies, 77 women received ≥1 INSTI (54 DTG, 14 elvitegravir, 15 raltegravir), 167 received non-INSTI, and 3 had missing data. Fifty congenital anomalies were identified in 36 infants. Infants with first-trimester DTG or any first-trimester INSTI exposure had higher odds of congenital anomalies than infants with first-trimester non-INSTI exposure (OR = 2.55; 95%CI = 1.07-6.10; OR = 2.61; 95%CI = 1.15-5.94, respectively). Infants with INSTI exposure after the second trimester had no increased odds of anomalies. Women with INSTI exposure had higher odds of preeclampsia (OR = 4.73; 95%CI = 1.70-13.19). Among women who received INSTI, grade ≥3 laboratory abnormalities were noted in 2.6% while receiving the INSTI and 3.9% while not receiving the INSTI, versus 16.2% in women who received non-INSTI. There was no association between INSTI exposure and other pregnancy outcomes.ConclusionIn our cohort, first-trimester INSTI exposure was associated with increased rates of congenital anomalies and use of INSTI during pregnancy was associated with preeclampsia. These findings underscore the need for continued monitoring of the safety of INSTI in pregnancy.

BackgroundMany women living with HIV (WLHIV) are co-infected with cytomegalovirus (CMV), Toxoplasma gondii (T gondii), and/or hepatitis C virus (HCV). The rates of congenital or perinatal transmission of these co-infections are not well defined in the current era, when most WLHIV receive antiretroviral therapy (ART) during pregnancy.MethodsRetrospective review of infants of WLHIV born between 2009–2019. Mothers were screened for antibodies to CMV, T. gondii, and HCV; chronic HCV infection was confirmed using plasma RNA PCR. Infants whose mothers had positive/unknown serostatus were screened for CMV using urine or saliva DNA PCR or culture at ≤3 weeks of life; T. gondii using serology at ≤1 month; and HCV using plasma RNA PCR at ≤6 months and serology at ≥12 months.ResultsThe study included 264 infants from 255 pregnancies in 191 mothers. At delivery, the median (IQR) CD4 count was 569 (406–748) cells/mm3 and plasma HIV load was 0 (0–24) RNA copies/mL. Among 243 infants born to CMV-seropositive (209) or CMV-missed serostatus (25) mothers, 163 (67.1%) were tested for CMV. Four infants had CMV detected, resulting in a rate of congenital infection of 2.5%. Among 65 infants from 54 (21.2%) pregnancies in T. gondii-seropositive women and 8 in women with unknown T. gondii-serostatus, one acquired congenital toxoplasmosis in the setting of acute maternal T. gondii infection. There were no episodes of vertical transmission from mothers with latent toxoplasmosis. Among 18 infants from 13 (5.1%) pregnancies in HCV RNA PCR-positive women and 4 in women with unknown HCV serostatus, there were no congenital or perinatal HCV transmissions.ConclusionsIn a US cohort of pregnant WLHIV on ART, we identified high maternal CMV seroprevalence and a high rate of congenital CMV infection. We did not identify any congenital or perinatal transmissions of T. gondii or HCV among mothers who had latent or chronic infections. Our data support screening pregnant WLHIV and their infants for CMV and suggest that the rates of congenital and perinatal T. gondii and HCV infections among infants born to WLHIV on ART may be lower in the era of effective ART.

Climate change impacts have become a verifiable reality in most communities in Africa and have already shown its ruthlessness in derailing modest gains made toward sustainable development. While evidence of climate change impacts abounds, especially in key climate-sensitive sectors, not many people living in affected communities have the requisite knowledge, understanding and capacity to respond to emerging impacts. Most communities in Ghana and Africa, broadly, lack the requisite climate change knowledge resources to inform adaptation choices. Adaptation decision-making, in most cases, is reactive, speculative, and based on flawed assumptions and understandings of the climate change phenomenon. This is essentially because most countries lack the capacity to make climate-informed decisions which is also a function of the pervasive lack of efficient climate information services regime across Africa. The paucity of climate change knowledge and associated climate information services is undoubtedly an issue of institutional capacity; however, it is also a function of an enduring culture—a poor attitude toward data collection and application—in decision-making processes. Data-poor environment, or data-poverty, as implied in this work, therefore, broadly describes the absence of a data management culture in decision-making processes; however, specifically to climate change, it describes the lack of functional climate information services regime in local communities in Africa and how such omissions impede the ability of countries to make climate-informed decisions to support adaptation and resilience building. Focusing on Ghana, the paper problematizes the lack of climate information in local communities. The paper argues that Africa's climate crisis is as much a knowledge and learning challenge which requires new and innovative learning approaches to build capacities to facilitate the making of data-driven and climate-informed adaptation decisions in local communities. The paper, therefore, foregrounds citizen-science networks as avenues for community-focused and community-based climate knowledge co-producing mechanisms.

The POLISH2 optical polarimeter has been in operation at the Lick Observatory 3 m Shane telescope since 2011, and it was commissioned at the Gemini North 8 m in 2016. This instrument primarily targets exoplanets, asteroids, and the Crab Pulsar, but it has also been used for a wide variety of planetary, galactic, and supernova science. POLISH2's photoelastic modulators, employed instead of rotating wave plates or ferroelectric liquid crystal modulators, offer the unprecedented ability to achieve sensitivity and accuracy of order 1 ppm (0.0001%), which are difficult to obtain with conventional polarimeters. Additionally, POLISH2 simultaneously measures the intensity (Stokes I ), linear polarization (Stokes Q and U ), and circular polarization (Stokes V ), which fully describe the polarization state of incident light. We document our laboratory and on-sky calibration methodology and our archival on-sky database, and we demonstrate the conclusive detection of circular polarization of certain objects.

Objectives: Diet-driven obesity is increasingly widespread. Its consequences pose major challenges to human health and health care systems. There are MAP kinase-interacting kinases (MNKs) in mice, MNK1 and MNK2. Studies have demonstrated that mice lacking either MNK1 or MNK2 were partially protected against high-fat diet (HFD)-induced weight gain and insulin resistance. The aims of this study were to evaluate the phenotype of mice lacking both MNKs when given an HFD, to assess whether pharmacological inhibition of MNK function also protects against diet-induced obesity (DIO) and its consequences and to probe the mechanisms underlying such protection. Methods: Male wild-type (WT) C57Bl6 mice or mice lacking both MNK1 and MNK2 (double knockout, DKO) were fed an HFD or control diet (CD) for up to 16 weeks.In a separate study, WT mice were also given an HFD for 6 weeks, after which half were treated with the recently-developed MNK inhibitor ETC-206 daily for 10 more weeks while continuing an HFD. Metabolites and other parameters were measured, and the expression of selected mRNAs and proteins was assessed. Results: MNK-DKO mice were almost completely protected from HFD-induced obesity. Higher energy expenditure (EE) in MNK-DKO mice was observed, which probably reflects the changes in a number of genes or proteins linked to lipolysis, mitochondrial function/biogenesis, oxidative metabolism, and/or ATP consumption. The MNK inhibitor ETC-206 also prevented HFD-induced weight gain, confirming that the activity of the MNKs facilitates weight gain due to excessive caloric consumption. Conclusions: Disabling MNKs in mice, either genetically or pharmacologically, strongly prevents weight gain on a calorie-rich diet. This finding likely results from increased energy utilisation, involving greater ATP consumption, mitochondrial oxidative metabolism, and other processes.

Reduced insulin-like signaling is required for C. elegans response to many environmental stressors, but how distinct outcomes are achieved is unknown. The authors show that the cytosolic sulfotransferase SSU-1 controls neurohormonal signaling via NHR-1 to specify the animals’ osmotic stress response.

Aim: The present study investigates the role of NF-κB in Bmpr2-related pulmonary hypertension (PH) using a murine model of PH with inducible overexpression of a cytoplasmic tail Bmpr2 mutation. Methods and Results: Electrophoretic mobility shift assay for nuclear extracts in Bmpr2R899X mouse lung and immunohistochemistry for NF-κB p65 in human PAH lung demonstrate that NF-κB is activated in end-stage disease. Acute inflammation or expression of a constitutively active NF-κB elicits a strong suppression of the BMP pathway in mice inversely correlating to activation of NF-κB targets. However, Bmpr2 mutation does not result in NF-κB activation in early disease development as assessed by luciferase reporter mice. Moreover, Bmpr2 mutant mice in which NF-κB activation is genetically blocked develop PH indistinguishable from that without the block. Finally, delivery of a virus causing NF-κB activation strongly exacerbates development of PH in Bmpr2 mutant mice, associated with increased remodeling. Conclusion: NF-κB activation exacerbates, but is not required for Bmpr2-related PH. Pulmonary vascular-specific activation of NF-κB may be a “second hit” that drives penetrance in heritable PH.

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA-driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans.

Anti-NeuN (Neuronal Nuclei) is a monoclonal antibody used extensively to specifically detect post-mitotic neurons. Anti-NeuN reactivity is predominantly nuclear; by western it detects multiple bands ranging in molecular weight from 45 kDa to >75 kDa. Expression screening putatively identified R3hdm2 as NeuN; however immunoprecipitation and mass spectrometry of the two major NeuN species at 45-50 kDa identified both as the RNA binding protein Rbfox3 (a member of the Fox family of alternative splicing factors), confirming and extending the identification of the 45 kDa band as Rbfox3 by Kim et al. Mapping of the anti-NeuN reactive epitopes in both R3hdm2 and Rbfox3 reveals a common proline- and glutamine-rich domain that lies at the N-terminus of the Rbfox3 protein. Our data suggests that alternative splicing of the Rbfox3 pre-mRNA itself leads to the production of four protein isoforms that migrate in the 45-50 kDa range, and that one of these splicing choices regulates Rbfox3/NeuN sub-cellular steady-state distribution, through the addition or removal of a short C-terminal extension containing the second half of a bipartite hydrophobic proline-tyrosine nuclear localization signal. Rbfox3 regulates alternative splicing of the Rbfox2 pre-mRNA, producing a message encoding a dominant negative form of the Rbfox2 protein. We show here that nuclear Rbfox3 isoforms can also enhance the inclusion of cryptic exons in the Rbfox2 mRNA, resulting in nonsense-mediated decay of the message, thereby contributing to the negative regulation of Rbfox2 by Rbfox3 through a novel mechanism.

The ability of germ cells to carry out a gene regulatory program distinct from the surrounding somatic tissue, and their capacity to specify an entire new organism has made them a focus of many studies that seek to understand how specific regulatory mechanisms, particularly post-transcriptional mechanisms, contribute to cell fate. In zebrafish, germ cells are specified through the inheritance of cytoplasmic determinants, termed the germ plasm, which contains a number of maternal mRNAs and proteins. Investigation of several of these messages has revealed that the restricted localisation of these mRNAs to the germ plasm and subsequent germ cells is due to cis-acting sequence elements present in their 3'UTRs. Here we show that a member of the Hu family of RNA-binding proteins, HuB, is maternally provided in the zebrafish embryo and exhibits germ cell specific expression during embryogenesis. Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3'UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells. In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3'UTR. Our study suggests that DAZL specifically binds the HuB 3'UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein.

Accurate determinations of individuals' one-repetition maximum (1RM) are critical when evaluating the effectiveness of an exercise intervention involving progressive resistance training (RT). Traditional ("bottom-up"; BT) testing methods involve progressions from low to maximal loads and are commonly used in clinical and laboratory environments. Concerns about the reliability of BT testing in certain populations suggest a different technique may be more effective. Purpose: To compare the reliability and effectiveness of traditional 1RM testing to a novel technique (TDT) involving progressive load reductions and a starting intensity equal to 130% of exercisers' estimated 1RM. Method: 70 healthy adults (age = 45.03 ± 25.64 y) with diverse RT experience were randomized into a reliability testing trial (RTT; n = 33) or an optimal method trial (OMT; n = 37). Subjects in the RTT performed either TDT or BT on 3 occasions separated by = 72 hours, while subjects in the OMT performed each method once in random order on separate days. Results: No significant differences in percent coefficient of variation were observed between BT and TDT for either exercise used in the study (pneumatic seated chest press: Hedge's g = 0.25, p = 0.49; pneumatic recumbent leg press: Hedge's g = 0.12, p = 0.74). TDT was not found to produce significantly higher 1RM values than BT in any group. Conclusion: TDT does not appear to facilitate more reliable 1RM estimates than BT. Further research is needed to determine the stability of these findings across levels of exercisers' age, sex, and previous RT experience.

Background: Patients with ulcerative colitis (UC) who are hospitalised for acute severe flares represent a high‐risk orphan population. Aim: To provide guidance for clinical trial design methodology in these patients. Methods: We created a multi‐centre consortium to design and conduct a clinical trial for a novel therapeutic intervention (hyperbaric oxygen therapy) in patients with UC hospitalised for moderate–severe flares. During planning, we identified and addressed specific gaps for inclusion/exclusion criteria; disease activity measures; pragmatic trial design considerations within care pathways for hospitalised patients; standardisation of care delivery; primary and secondary outcomes; and sample size and statistical analysis approaches. Results: The Truelove‐Witt criteria should not be used in isolation. Endoscopy is critical for defining eligible populations. Patient‐reported outcomes should include rectal bleeding and stool frequency, with secondary measurement of urgency and nocturnal bowel movements. Trial design needs to be tailored to care pathways, with early intervention focused on replacing and/or optimising responsiveness to steroids and later interventions focused on testing novel rescue agents or strategies. The PRECIS‐2 framework offers a means of tailoring to local populations. We provide standardisation of baseline testing, venous thromboprophylaxis, steroid dosing, discharge criteria and post‐discharge follow‐up to avoid confounding by usual care variability. Statistical considerations are provided given the small clinical trial nature of this population. Conclusion: We provide an outline for framework decisions made for the hyperbaric oxygen trial in patients hospitalised for UC flares. Future research should focus on the remaining gaps identified. [ABSTRACT FROM AUTHOR]

Literature on the drivers of flooding in sub-Saharan Africa is mostly focused on large and megacities, despite the majority of cities in the region being medium- and small-sized. We conducted a systematic review using 27 peer-reviewed scientific papers selected following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocols using the Greater Kumasi Metropolitan Area (GKMA), Ghana, as a case study, and validated the results in a workshop attended by 51 stakeholders. We found that the reported drivers of flood risk in GKMA are land cover changes (61.5%) (e.g. wetlands degradation); governance challenges (16.2%); challenges with drainage (13%); extreme rainfall (6.5%); and apathy towards nature (3.2%). We conclude that floods in GKMA are overwhelmingly human induced through unsustainable urbanization processes that are so rapid that city authorities are unable to properly control them. Also, a holistic understanding of flood risk is missing since majority of the included papers rather used qualitative methodologies. [ABSTRACT FROM AUTHOR]

When reviewing the safety of food packaging, safety assessors must determine the migration levels of a food contact substance (FCS) from the packaging into the contained food products. Migration values can be expressed relative to levels in food using a single 'food mass-to-surface area (mF/A) ratio', representative of the general food supply. Because no published literature has examined mF/A ratios for the current U.S. food supply, this study aimed to use food databases to develop a standard mF/A ratio representative of packaged foods in the current U.S. food supply. First, we obtained information on the food-contact surface area (A) and food mass (mF) for a selection of packaged foods sold in U.S. retail stores. To adequately represent the variation of food products on the market, we identified a representative product per food category, using a 95-category list that was derived from the USDA WWEIA Food Categories. The values for A and mF per package were determined for each representative product using product size information from the Syndigo Nutrition Database. These data were used to calculate the A/mF ratio for each of the 95 representative products, and the average A/mF ratio was determined. The average A/mF ratio was weighted using consumption data from the WWEIA component of NHANES, so that more weight was given to foods that are consumed in greater quantities. The final step was to then take the reciprocal of the averaged A/mF ratio to obtain the final mF/A ratio. The resulting mF/A ratio was 4.8 g/in², which rounds to 5 g/in². In summary, we determined that 5 g/in² is representative of the current U.S. food supply and could be used as a standard mF/A ratio when estimating migration levels of an FCS from packaging into food. [ABSTRACT FROM AUTHOR]

Near-wall mass transport plays an important role in many cardiovascular processes, including the initiation of atherosclerosis, endothelial cell vasoregulation, and thrombogenesis. These problems are characterized by large Péclet and Schmidt numbers as well as a wide range of spatial and temporal scales, all of which impose computational difficulties. In this work, we develop an analytical relationship between the flow field and near-wall mass transport for high-Schmidt-number flows. This allows for the development of a wall-shear-stress-driven transport equation that lies on a codimension-one vessel-wall surface, significantly reducing computational cost in solving the transport problem. Separate versions of this equation are developed for the reaction-rate-limited and transport-limited cases, and numerical results in an idealized abdominal aortic aneurysm are compared to those obtained by solving the full transport equations over the entire domain. The reaction-rate-limited model matches the expected results well. The transport-limited model is accurate in the developed flow regions, but overpredicts wall flux at entry regions and reattachment points in the flow.

Intraluminal thrombus (ILT) in abdominal aortic aneurysms (AAA) has potential implications to aneurysm growth and rupture risk; yet, the mechanisms underlying its development remain poorly understood. Some researchers have proposed that ILT development may be driven by biomechanical platelet activation within the AAA, followed by adhesion in regions of low wall shear stress. Studies have investigated wall shear stress levels within AAA, but platelet activation potential (AP) has not been quantified. In this study, patient-specific computational fluid dynamic (CFD) models were used to analyze stress-induced AP within AAA under rest and exercise flow conditions. The analysis was conducted using Lagrangian particle-based and Eulerian continuum-based approaches, and the results were compared. Results indicated that biomechanical platelet activation is unlikely to play a significant role for the conditions considered. No consistent trend was observed in comparing rest and exercise conditions, but the functional dependence of AP on stress magnitude and exposure time can have a large impact on absolute levels of anticipated platelet AP. The Lagrangian method obtained higher peak AP values, although this difference was limited to a small percentage of particles that falls below reported levels of physiologic background platelet activation.

[no abstract]